The purpose of these studies is to analyze the molecular mechanisms by which insulin-like growth factor-l (IGF-I) stimulates smooth muscle cell (SMC) migration and replication. SMC synthesize IGF binding protein -4 (IGFBP-4) and an IGFBP-4 protease. IGFBP-4 inhibits IGF-I binding to receptors and the protease facilitates its release. These studies will focus on expressing pure fibulin 1-Cwhich has IGFBP-4 protease activity, identifying the factors that regulate its synthesis and activation and determining its physiologic role in releasing IGF-I to SMC. A protease resistant form of IGFBP-4 will be used to assess the importance of release of IGF-I into the pericellular space for atherosclerotic lesion development. Several intergral membrane proteins regulate the ability of target cells such as SMC to respond to IGF-I. These include the IGF-I receptor, the aV(33 integrin/integrin associated protein (IAP) and SHPS-1. To study the interaction between IAP and aV|33 we will prepare IAP mutants that do not bind to aVp3, express them in SMC and determine if cells expressing these mutants have an altered biologic response to IGF-I. Since truncation of aV alters aV|33 binding to IAP we will utilize cells expressinga truncated aV mutant to determine how this alters IGF-I stimulated binding to IAP. Since changes in IAP binding within lipid domains of cell membranes are important for controlling whether it binds to aV(33 we will determine how IGF-I facilitates this process Atheroslerotic lesions will be analyzed to determine if IGF-I stimulates the association of IAP with aVp3 in vivo. To determine how ligand occupancy of IAP modulates cellular responsiveness to IGF-I, we will prepare an IAP mutant that cannot bind to its principle ligand thrombospondin-1 (TSP-1) and determine if cells that express this mutant have altered biologic responsesto IGF-I. We will analyze the mechanism by which TSP-1 binding to IAP is altering IGF receptor function and determine if TSP-1 binding to IAP is functioning through SHPS-1 to alter the rate at which the IGF-I receptor is dephosphorylated. The results of these studies should define multiple new molecular mechanisms by which IGF-I functions coordinately with extracellular matrix proteins to activate its receptor and stimulate SMC replication and migration. The results may suggest novel strategies for interfering with these processes to alter the progression of atherosclerosis.